Distinct Allelic Patterns of Nanog Expression Impart Embryonic Stem Cell Population Heterogeneity

Abstract

Nanog is a principal pluripotency regulator exhibiting a disperse distribution within stem cell populations in vivo and in vitro. Increasing evidence points to a functional role of Nanog heterogeneity on stem cell fate decisions. Allelic control of Nanog gene expression was reported recently in mouse embryonic stem cells. To better understand how this mode of regulation influences the observed heterogeneity of NANOG in stem cell populations, we assembled a multiscale stochastic population balance equation framework. In addition to allelic control, gene expression noise and random partitioning at cell division were considered. As a result of allelic Nanog expression, the distribution of Nanog exhibited three distinct states but when combined with transcriptional noise the profile became bimodal. Regardless of their allelic expression pattern, initially uniform populations of stem cells gave rise to the same Nanog heterogeneity within ten cell cycles. Depletion of NANOG content in cells switching off both gene alleles was slower than the accumulation of intracellular NANOG after cells turned on at least one of their Nanog gene copies pointing to Nanog state-dependent dynamics. Allelic transcription of Nanog also raises issues regarding the use of stem cell lines with reporter genes knocked in a single allelic locus. Indeed, significant divergence was observed in the reporter and native protein profiles depending on the difference in their half-lives and insertion of the reporter gene in one or both alleles. In stem cell populations with restricted Nanog expression, allelic regulation facilitates the maintenance of fractions of self-renewing cells with sufficient Nanog content to prevent aberrant loss of pluripotency. Our findings underline the role of allelic control of Nanog expression as a prime determinant of stem cell population heterogeneity and warrant further investigation in the contexts of stem cell specification and cell reprogramming.

Author Summary

Nanog is a key factor influencing the decision of a stem cell to remain pluripotent or differentiate. Each embryonic stem cell (ESC) in a population exhibits fluctuating Nanog levels resulting in heterogeneity which affects cell fate specification. The allelic regulation of Nanog was demonstrated recently but its implications on population heterogeneity are unclear. We developed a multiscale population balance equation (PBE) model and compared our results with pertinent experimental studies. Under allelic control the profile of Nanog features three peaks or distinct states. Transcriptional noise causes the distribution to become bimodal as suggested previously. When stem cells carrying a reporter transgene in an allelically regulated locus were examined, we observed non-matching distributions of the endogenous and reporter proteins. This led us to investigate the performance of reporter systems depending on insertion of the transgene in one or both alleles and the protein degradation dynamics. Lastly, our model was employed to address how allelic regulation affects the maintenance of pluripotency in stem cells with a single Nanog allele deletion. A fraction of these cells remains pluripotent while deletion of a single allele does not simply reduce NANOG uniformly for all ESCs but modulates NANOG heterogeneity directly.